Possible mechanisms underlying the involvement of microglial P2X4 receptors in the comorbidity of pain and depression

doi:10.21203/rs.3.rs-4721040/v1

Download PDF

Short Report

Possible mechanisms underlying the involvement of microglial P2X4 receptors in the comorbidity of pain and depression

https://doi.org/10.21203/rs.3.rs-4721040/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Microglia’s P2X4 receptor may be a key target involved in the occurrence and development of pain–depression comorbidity. The objective of this study is to explore the possible mechanisms underlying the involvement of microglial P2X4 receptors in pain–depression comorbidity. Sixty male SD rats were randomly divided into six groups: normal control group (10), sham operation group (10), chronic constriction injury of sciatic nerve group (CCI group, 10), CCI + antagonist group (10), chronic unpredicted mild stress model group (CUMS group, 10), CUMS + antagonist group (10). The normal group received no treatment, the sham group was injected with normal saline through stereotaxic localisation, the CCI group was ligated with sciatic nerve of left leg, the CUMS group was stimulated by stress for 30 days. The rats in the CCI group and CUMS group were injected with P2X4 receptor antagonist 30 days later. The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured in each group on different days, followed by forced swimming tests in all groups to measure floating immobility time in water. Finally, P2X4’s expression in rostral ventromedial medulla (RVM) and spinal lumbosacral swelling were detected by Western Blotting and immunofluorescence, respectively, whilst expression of 5-HT in the spinal dorsal horn was measured via immunohistochemistry. Compared with the sham group, both MWT and TWL in the CCI group exhibited a decreasing trend (lowest level at approximately 2 weeks before remaining stable). Floating state time in the forced swimming experiment was significantly prolonged. Simultaneously, P2X4’s expression increased in RVM, as did that of 5-HT in spinal cord, but the expression of 5-HT in spinal cord significantly decreased after injection of P2X4 receptor antagonist in RVM. Compared with the normal group, MWT and TWL in the CUMS group decreased gradually, and floating state time reached the longest. P2X4 receptor expression in the RVM increased more significantly than that in the CCI group, and that of 5-HT in the spinal cord increased. Post injection of the P2X4 receptor antagonist, the MWT and TWL were higher than those before injection, and forced swimming time decreased. Moreover, P2X4 receptor expression in RVM increased, and expression of 5-HT in the spinal cord decreased. P2X4 receptor in RVM may be involved in the pathogenesis of pain–depression comorbidity.

chronic pain

P2X4 receptor

rostral ventromedial medulla

sciatic nerve ligation

chronic stress depression

Currently, patients with chronic pain often experience negative emotions, such as disgust, anxiety, fear and depression. Due to the increasing pressure of work and life, the proportion of people suffering from depression, or potential depression, is still extremely large, and there is an increasing trend year by year. Chronic pain, as a common stimulus factor of depression, is causing great trouble for, and placing burdens on, human beings^[1]. According to the latest clinical epidemiological data, 52% of patients with chronic pain have depressive symptoms, and 65% of depressed patients have pain manifestations^[2]. Therefore, the purpose of this study is to detect the mechanism in the common factors of pain and depression.

The comorbidity of pain and depression is very common, but the specific mechanism is very complicated. At present, some scholars believe that the mechanism of pain and depression comorbidity is mainly related to the notion that chronic pain leads to central and peripheral inflammatory reactions, and then causes the changes in the tryptophan pathway, hypothalamic-pituitary-adrenal axis hyperactivity, oxidation/nitrification pathway activation and other reactions, before finally leading to the occurrence of pain and depression comorbidity^[3]. Recent studies have shown that the output of nociceptive sensations in the dorsal horn of the spinal cord after peripheral nerve injury is not completely dependent on neurons, and the interaction between microglia and neurons plays a key role in generating and maintaining neuropathic pain, in which the key channel between microglia and neurons is the P2X4 receptor^[4]. Further experiments discovered that mice with the P2X4 receptor gene knocked out had significantly less mechanical hypersensitivity to pain after sciatic nerve ligation, whilst wild-type mice experienced the opposite effect^[5]. The activation of the P2X4 receptor plays a key role in the occurrence of mechanical hypersensitivity to pain after nerve injury, and the up-regulation of P2X4 receptor expression plays an important role in the prolongation and development of hypersensitivity to pain. Some researchers also believe that chronic pain reduces the expression of the P2X4 receptor in the tryptophan pathway under negative feedback regulation, resulting in depression^[6]. However, the way in which the tryptophan pathway works is still unclear. It is currently believed that, during nerve injury, primary sensory neurons, spinal dorsal horn neurons, or spinal dorsal horn astrocytes release a large amount of ATP, activate microglia, lead to the 5-HT receptor becoming up-regulated, and cause Ca²⁺ internal flowing, thus mediating protein kinase C and translocating Ca²⁺ from the cytoplasm to the cell membrane, whilst also releasing diglycerol and Ca²⁺ activate protein kinase C together^[7]. The activated protein kinase C then transfers to the nucleus, phosphorylates transcription factors, promotes the proliferation of microglia, and affects the process of pain sensitisation. Therefore, in this study, the CCI model and CUMS model were established to detect the expression of the P2X4 receptor in the RVM and 5-HT in the spinal cord, as well as to explore whether the P2X4 receptor is involved in the occurrence of pain and depression comorbidity.

1.Ethics statement

All animal experiments were approved by the Ethics Committee of the Wuxi People’s Hospital (approval number: MS201916).

2.Materials and instruments

The materials and instruments employed were as follows: Rabbit polyclonal P2X4 Antibody, Sheep Anti-Rabbit Antibody (sigma), Rabbit Anti-rat P2X4 Antibody, P2X4 receptor antagonist (5-BDBD) (sigma), pain behaviour measuring instrument (MWT/TWL) (Life Science Corporation of the United States), rat brain localisation instrument (Shanghai Xinsoft Company), laser confocal microscope (Leica Company of Germany), frozen microtome (Leica Company of Germany), forced swimming experimental device and video monitoring software (Shanghai Xinsoft Company).

3. Experimental animals and grouping

A total of 60 clean male SD rats, weighing approximately 250 g, were provided by Slack Animal Research Center, Shanghai. Using the random number table method, the rats were divided into six groups: normal control group (10), sham operation group (10), chronic constriction injury of sciatic nerve ligation group (CCI group, 10), CCI + antagonist group (10), chronic unpredicted mild stress of depression model group (CUMS group, 10), and the CUMS + antagonist group (10). The normal group did not receive any special treatment, and instead only normal feeding. The CUMS group was treated with one of seven stress factors, including fasting, water prohibition, day and night reversal, tail clip for 1 min, 0 ℃ ice water bath for 5 min, 40 ℃ hot water bath for 5 min, and tilting of the cage. One or two stress factors were randomly applied every day. The same stress factor appeared discontinuously, which made the mice unpredictable for 1 month. The P2X4 receptor antagonist group involved the administering of a stereotaxic injection into the brains of the rats. The dose of the antagonist was 0.1 mg/kg, which was added to 1 ul normal saline and injected with 2 ul microsyringe for 5 min. The coordinates were bregma (-10.5 mm, 0 mm, -8.5 mm) in the RVM region of the brain.

The mechanical hypersensitivity threshold (MWT) and heat shrinkable claw latency (TWL) of each group were measured, following which forced swimming experiments were carried out in each group. The floating immobile time of rats in warm water around 28 ℃ within 5 min was measured using video software. After the behavioural test, the RVM and lumbosacral swelling of the spinal cord were selected, and the expression of the P2X4 receptor in RVM was detected using Western Blotting and immunofluorescence, respectively, whilst the expression of 5-HT in the dorsal horn of the spinal cord was measured via immunohistochemistry.

All animal experiments were approved by the Ethics Committee of the Wuxi People’s Hospital.

4. Methods and procedures

(1) Establishment of CCI model

The rats were first weighed and then intraperitoneally injected with 3% pentobarbital sodium at 30 mg/kg. In lateral position, the left hind limb was selected on the surgical side. Before surgery, the skin was disinfected and covered with a hair removal agent. The skin was cut approximately 1 cm below the femur parallel to the femur, and the muscular fascia was bluntly separated with a small peel to fully expose the main sciatic nerve. The catgut was wrapped around the sciatic nerve for single knot fixation, and three knots were tied in succession; each knot was around 1 cm away, subject to a slight twitch of the first knot muscle. The tissue and skin were then sutured after saline irrigation, and a small amount of 2% lidocaine was applied to the wound with a cotton swab. After the operation, the rats were placed in a cage and fed in a free environment^[8].

(2) Establishment of CUMS model

The establishment of the CUMS model is mainly based on the description in the literature, and subsequently the operation is carried out according to the environmental conditions of the laboratory^[9]. During the experiment, common unpredictable stress stimuli were selected, such as fasting, water prohibition (24 h), squirrel cage tilt, wet cage, etc. In order to make the stress have obvious unpredictability, the stress stimulus was selected at any time during the day and night, and the stress conditions were randomly chosen; one or two kinds of stress were selected every day, and the same stress stimulus did not appear continuously. The whole process of creating the model continued for 30 days without interruption, and finally a forced swimming experiment was carried out for detection.

(3) Thermal withdrawal latency (TWL)

The incubation period of plantar heat pain response was measured using a thermal radiation stimulator. The rats were placed in a clean plexiglass box at 8 a.m. every day and covered with a baffle to acclimate them for 1 hour. Following this, the soles of the rats’ feet were irradiated with the thermal radiation stimulator. The incubation period from the beginning of irradiation to the appearance of leg lifting avoidance was the thermal foot retraction reflex. The intensity of thermal stimulation was adjusted to the basic value of 12 to 15 s, and was maintained consistently throughout the experiment. The automatic cut-off time was 25 s to avoid thermal radiation damage. Moreover, in order to avoid or reduce the influence of the previous stimulus on the subsequent stimulus effect, the interval time of stimulation at the same site was 10 min, and the recording time was measured continuously at least three times.

(4) Mechanical withdrawal threshold (MWT)

Using an electronic pain meter, the measurement time was fixed at 10 a.m., at 7, 14, and 30 days, whilst the indoor temperature was controlled at 24 ℃. In a quiet and bright laboratory, the rats were placed on a metal mesh shelf with a transparent cover, approximately 30 cm away from the tabletop. After adjusting to the environment for 45 min, the rats were calm and measured. The stimulation site was the same in the experimental group and the reference group. The maximum value of the electronic pain meter was recorded after the foot withdrawal reaction of the rats, and the average value was measured three times when the rats were in their calm state. The measurement time point was the same as the TWL value.

(5) Forced swim test

The purpose of this experiment was to test depression in rats. The experiment was conducted at night when the rat activity was normal, and the light was dim during the experiment. Moreover, the observer was kept a certain distance from the experimental animal so as to avoid affecting the behaviour of that animal. First, the transparent cylindrical (height: 100 cm, diameter: 20 cm) glass container was injected with 30 cm deep water; the water temperature was maintained at approximately 25°C, and the room temperature was maintained at 23°C. Each rat was tested separately, and 24 h before the formal test, the rats were pre-swam for 15 min to familiarise them with the water and the water temperature. During the test, the rats were gently placed in water, and the rats were first adapted to swimming for 1 min, whilst the sum of the floating immobile time of the rats in the following 5 min was recorded from the second minute. The total time of each forced swimming experiment was 6 min. After the experiment, the rats were quickly removed from the water, their hair dried, and gently placed back in the cage^[10].

5. Immunohistochemical fluorescence detection

Three rats in each group were randomly selected, anaesthetised with 3% pentobarbital sodium, and underwent rapid thoracotomy for intracardiac perfusion. Lumbosacral enlargement (L4-L6) was obtained, then fixed, dehydrated, and sliced.

Immunohistochemical procedure: Add 3% H202 to eliminate endogenous peroxidase for 10 min; add rabbit-derived rat 5-HT (1:300) primary antibody, and incubate at 4 ℃ for 24 h. The second antibody was incubated using the rabbit hypersensitive immunohistochemical three-step kit instructions: DAB colour development, patch, alcohol gradient dehydration, xylene transparent, neutral gum seal, and photo.

Immunofluorescence procedure: 0.3%Teiton X-100 permeated the membrane for 30 min and was closed with protein dry powder. 1:300 primary antibody of c-fos was incubated at 4 ℃ overnight. Fluorescent secondary antibodies (FITC-labelled Goat Anti-Rabbit IgG /FITC 1:000) were added and incubated at room temperature for 2 h, in the dark. The patch, the anti-fluorescence quenching sealing plate, and the liquid sealing plate were observed and photographed under a fluorescence microscope.

6. Immunowestern blot detection

All of the rats were killed using an intraperitoneal injection of 3% pentobarbital sodium, and the brains were quickly decapitated. The ventral part of the cerebellum was separated on ice and placed in an EP tube. Tissue lysate and an enzyme inhibitor were added; the homogenate was homogenised on ice, the supernatant was taken, the protein content was measured using the BCA method, and the protein loading buffer (5×) was added to SDS-PAGE. Gel was prepared according to the instructions, whilst the marker and sample were added and placed in the electrophoresis device until bromophenol blue reached the bottom of the separation glue. The gel was removed and the membrane rotated. After the end of membrane transfer, the protein was sealed with dry powder for 2 h. Incubated mice were treated with the rat P2X4 receptor primary antibody (1:500 dilution) and the GAPDH primary antibody (1:500 dilution) at 4 ℃ overnight. The sheep anti-rabbit IgG secondary antibody (1:1,000) was labeled with alkaline phosphatase and incubated at room temperature for 2 h. Subsequently, NBT/BCIP chromogenic reagent was added for colour development. Finally, the strips were taken out and scanned. Image J image analysis software was used to analyse the gray scale of the protein bands, and the gray scale values of each group were calculated. The ratio of the gray scale of the target protein group to that of the internal reference group (GAPDH) was used to represent the relative expression level of the target protein.

7. Statistical analysis

Graph PAD 8.0 statistical software was employed for data entry and statistical analysis, and statistical charts were generated. All measurement data were expressed as mean ± standard deviation (\(\:\stackrel{-}{x}\pm\:\)SD). A t-test was used for comparison between the two groups, and a paired t-test was employed for comparison before and after. One-way ANOVA was employed for multi-group comparison, and the TUKEY test was utilised for pairwise comparison. P<0.05 was considered statistically significant.

1. Changes of pain threshold in each group

The basic MWT values of the selected rats were similar, and there was no significant difference amongst them. Compared with the sham group, the threshold value in the CCI group decreased significantly (P < 0.001) after 7 and 14 days, and then remained relatively stable. At the same time, compared with the normal group, the threshold value of the CUMS group decreased significantly after 30 days (P < 0.05), suggesting that depression easily caused hyperalgesia. There was no significant difference between the normal group and the sham group at each time point. The TWL measurements were similar to the MWT measurements (Fig. 1).

In the CCI + antagonist group and CUMS + antagonist groups, an intracranial localisation injection of antagonist was given to measure MWT and TWL. MWT and TWL were higher compared to before the injection of antagonist in the CCI + antagonist group and CUMS + antagonist groups (P < 0.05). Moreover, TWL was much higher compared to before the injection of antagonist in the CUMS + antagonist group (P < 0.001, Fig. 2).

2. Forced swimming test results

During the 5-min forced swimming experiment, the software of the forced swimming system tested three states: floating state, swimming state and struggling state. On the day before the test, the rats were subjected to a 15-min pre-swimming experiment to familiarise them with water. The data showed that the swimming time of the normal group was significantly longer than that of other groups, and the immobile time was the shortest. Compared with the normal group, the floating immobile time of the CUMS group was significantly extended (P < 0.01), and the floating time of the CCI group was relatively extended compared with the sham group (P < 0.05). After the injection of antagonists, the floating time of the CCI + antagonists was shortened compared with that of the CCI group (P < 0.05), and the floating time of the CUMS + antagonists was also significantly reduced compared with that of the CUMS group (P < 0.01), suggesting that long-term chronic pain leads to depression (Table 1).

Table 1

Comparison of behaviour of rats in forced swimming test
Group	n	Floating time(S)	Swimming condition time(S)
Normal	10	333.46 ± 16.98	266.48 ± 18.45
Sham	10	35.64 ± 16.54	255.84 ± 21.58
CCI	10	136.59 ± 18.88^*	160.31 ± 19.21
CCI + antagonist	10	36.18 ± 10.98^#	365.55 ± 12.33
CUMS	10	241.57 ± 30.81^**	58.99 ± 29.67
CUMS + antagonist	10	88.67 ± 23.16^&&	215.89 ± 21.79
P < 0.05, * P < 0.01, vs. Normal group and Sham group; #P < 0.05, vs. CCI group; && P < 0.01, vs. CUMS group.

3. Detection of cell membrane receptors (P2X4 receptors) in RVM

P2X4 receptors in RVM are mainly synthesised and released by descending fibres in the brain stem, and P2X4 receptors are divided into P2X1, P2X2, P2X3 and other receptors, amongst which P2X4 receptors are closely related to depression. Western Blotting analysis showed that, compared with the normal and sham groups, the expression of the P2X4 receptor in RVM in the CCI group increased (P < 0.05), and the expression of P2X4 in RVM in the CUMS group increased more significantly (P < 0.01). Immunofluorescence results showed that the expression of the P2X4 receptor in RVM in the CCI group was significantly higher than that in the normal group, but less than that in the CUMS group, and the corresponding antagonist group was significantly reduced (P < 0.001) (Fig. 3).

4. Expression of 5-HT in dorsal horn of spinal cord

5-HT is the main neurotransmitter transmitting nociceptive information in the descending regulatory pathway. It is believed that the degree of pain is positively correlated with the expression of 5-HT in the spinal cord. Compared with the normal group, the expression of 5-HT in the spinal cord of the CCI group increased (P < 0.01), and the expression of 5-HT in the spinal cord of the CUMS group also increased (P < 0.05). This suggests that the increased expression of the 5-HT receptor in the two groups is transmitted to the spinal cord through the neurotransmitter 5-HT under the influence of descending regulatory factors, resulting in the increased expression of 5-HT in the spinal cord, which leads to the formation of hyperalgesia. After the other two groups were injected with antagonists, the expression of 5-HT in the spinal cord decreased, which indirectly indicated that the descending factors were involved in the regulation (Fig. 4).

In order to enhance the study of comorbidity between chronic pain and depression, we introduced the CCI model related to pain and depression, as well as the CUMS model related to depression. Zhang Yuqiu et al. suggested that chronic pain caused by the CCI model is a combination of emotional reactions and pain perception^[11]. Studies have found that emotional reactions, such as anxiety and depression, start appearing in rats 14 days after sciatic nerve ligation, and can persist for approximately 3 months. This indicates that rats in the CCI model exhibit a state of comorbidity between pain and depression after 14 days. In the current experiment, forced swimming tests were conducted with rats in the CCI model group, revealing reduced pain threshold and increased floating time, in turn indicating manifestation of comorbidity between pain and depression^[12].

Recent studies have proposed that the rostral ventromedial medulla plays a crucial role in understanding the comorbidity between pain and depression. It primarily consists of the nucleus raphe magnus (NRM) along with its adjacent ventral reticular formation^[13]. Nociceptive information is transmitted from peripheral receptors to the spinal dorsal horn through dorsal root neurons when experiencing pain, then reaching the sensory cortex via the dorsal thalamus. Subsequently, the upper spinal cord regulates nociceptive information through the descending modulatory system^[14]. RVM serves as a key nucleus within this descending modulatory system whilst also having direct or indirect neural projections with the hippocampus, amygdala, and cingulate gyrus^[15]. Therefore, RVM was selected as the primary focus in this study to investigate comorbidity between pain and depression.

Regarding physiological mechanisms involved in the pathogenesis of comorbid conditions involving both pain and depression, these are highly complex, without any unified theory; Leonard et al.^[16] forwarded the belief that chronic pain can cause inflammatory reactions in the central and peripheral regions, and then activate the oxidative/nitrative stress pathway, leading to depression. Maes et al.^[17] opined that the hypothalamic-pituitary-adrenal axis was significantly hyperactive under the action of chronic pain and PICs, promoting the development of depression. Fukuhara et al.^[18] were of the opinion that chronic pain led to changes in brain-derived neurotrophic factors and disorders in nerve regeneration and repair, resulting in depression. Further, Kim et al.^[19] held that pain and depression comorbidity were related to the tryptophan metabolic pathway by changing the activities of indole-2,3-dioxygenase and tryptophan-2,3-dioxygenase. Studies have shown that formalin injected into the hind limbs of rats induced the inflammatory pain model; it was also observed that P2X4 receptor up-regulation in ipsilateral spinal dorsal horn microglia reached the highest level after a formalin injection into the rat’s hind limb on the 7th day, and returned to the original level on the 14th day; indeed, this fully demonstrates that the P2X4 receptor, in the process of inflammatory pain, plays a certain role, although the specific mechanism has not been elaborated on. However, recent studies have now found that the tissue inherent macrophages constitutively express P2X4 receptor at rest; when there is activation of the P2X4 receptor in macrophages, there is calcium ion inflow, and p38MAPK phosphorylation, thereby activating intracellular phospholipase A2 (PLA2) and releasing cyclooxygenase-dependent PGE2, thus increasing the excitability of sensory neurons and producing hyperalgesia^[20]. A wild rat paw injection of ATP-activated macrophages can induce hyperalgesia. In the P2X4 gene knockout mice, the same phenomenon was not observed. Other studies have shown that, after peripheral nerve injury in CCL21 gene knockout mice, an intrathecal injection of CCL21 can rapidly induce hyperalgesia and P2X4 receptor up-regulation on microglia, and no intrathecal injection of CCL21 can up-regulate the expression of the P2X4 receptor on microglia^[21]. Thus, whether the P2X4 receptor is involved in the comorbidity of pain and depression has not been reported. The results of this experiment showed that the expression of the P2X4 receptor in rats in the CCI group increased. After injecting P2X4 receptor antagonist into rats in the CCI group, the floating state time of those rats was reduced, that is, depression was relieved. At the same time, the pain threshold of the rats increased and pain was reduced, which negatively confirmed that the P2X4 receptor was involved in the occurrence of pain and depression comorbidity. Therefore, this experiment speculated that the overexpression of the P2X4 receptor in RVM led to increased binding with 5-HT, reduced 5-HT content in the synaptic gap, and finally led to the occurrence of pain and depression comorbidity through the change of the tryptophan metabolic pathway.

The results of this experiment also revealed that depression can affect pain. Ikeda et al.^[22] found, after placing terminal cancer patients in an environment with a lavender smell and conducting clinical experiments, that euphoria could reduce pain. It was also discovered that keeping animals in a lemon-smelling environment for 2 weeks could increase the pain threshold to thermal stimulation and reduce formalin-induced pain behaviour. If euphoria can reduce pain, do depression and other bad emotions aggravate pain? Fu et al. found that pain aversion could aggravate pain by means of conditioned position avoidance experiments and plantar injection of formalin. In this experiment, the expression of 5-HT in the spinal cord increased and the pain threshold decreased in the CUMS group. Previous studies have confirmed that, under pathological conditions, pain is positively correlated with 5-HT expression. Therefore, this experiment speculated that, when depression occurs, the expression of the P2X4 receptor increases, and under the negative feedback regulation, the release of 5-HT decreases. Then, under the action of the higher central cortex, through the influence of descending regulation factors or other factors, the expression of 5-HT in the spinal cord increases, resulting in the phenomenon of decreased threshold and hyperalgesia. On the contrary, under the action of antagonists, the release of 5-HT is inhibited and weakened, and under the action of the descending inhibition system, the expression of 5-HT in the spinal cord decreases, resulting in increased tolerance of pain stimulation.

In summary, we conclude that the P2X4 receptor in microglia is involved in the occurrence of pain and depression comorbidity. The mechanism may be affected by the stimulation of noxious information. The overexpression of the P2X4 receptor affects the metabolism of 5-HT nerve energy, ultimately leading to the occurrence of pain and depression comorbidity.

Ethics approval and consent to participate: All animal experiments were approved by the Ethics Committee of the Wuxi People’s Hospital.

Consent for publication:

Not applicable.

Competing interests

The authors declare no competing interests.

Authors' information

WW is the attending physician of the Pain Department of Wuxi People’s Hospital. ZFG is Head of the Pain Department of Wuxi People’s Hospital and has experience in the treatment of acute and chronic pain, as well as in the diagnosis and treatment of neuralgia, neck and lumbar pain.

Funding:

This study was supported by the Key Research and Development Projects of Science and Technology Plan of Jiangsu Province (grant number EB2022697) and the National Major Research and Development Plan (grant number 2023YFC2507105). The funding source had no involvement in the study design or conduct; collection, analysis, and interpretation of data; preparation, review, or approval of the manuscript; or decision to submit the manuscript for publication.

Author Contribution

ZFG: conception of the trial design, experimental guidance, drafting of original manuscript; WW: performed experiments and major contributor in writing the manuscript; LX: performed experiments; CXH: interpretation of data, data processing and software operation; HZS: performed experiments; SP: performed experiments; JLL: biochemical analyses, performed histological evaluations; all authors read and approved the final manuscript.

Acknowledgements

The authors sincerely appreciate the contributions of our team that led to the success of the study. We would like to thank Editage (www.editage.com) for English language editing.

Data Availability

Data supporting this study are included within the article and/or supporting materials

Kremer, M., Becker, L. J., Barrot, M. & Yalcin, I. How to study anxiety and depression in rodent models of chronic pain? Eur. J. Neurosci. 53, 236–270 (2021). PMID: 31985104.
Bonilla-Jaime, H. et al. Depression and pain: use of antidepressants. Curr. Neuropharmacol. 20, 384–402 (2022), PMID: 34151765, PMCID: PMC9413796.
Basu, T., Maguire, J. & Salpekar, J. A. Hypothalamic-pituitary-adrenal axis targets for the treatment of epilepsy. Neurosci. Lett. 746, 135618 (2021). PMID: 33429002.
Sophocleous, R. A., Ooi, L. & Sluyter, R. The P2X4 receptor: cellular and molecular characteristics of a promising neuroinflammatory target. Int. J. Mol. Sci. 23, 5739 (2022), PMID: 35628550, PMCID: PMC9147237.
Inoue, K. Role of the P2X4 receptor in neuropathic pain. Curr. Opin. Pharmacol. 47, 33–39 (2019). PMID: 30878800.
Khir, N. A. M., Noh, A. S. M., Shafin, N. & Ismail, C. A. N. Contribution of P2X4 receptor in pain associated with rheumatoid arthritis: a review. Purinergic Signal. 17, 201–213 (2021). PMID: 33594635, PMCID: PMC8155137.
Liu, Q. Q. et al. Role of 5-HT receptors in neuropathic pain: potential therapeutic implications. Pharmacol. Res. 159, 104949 (2020). PMID: 32464329.
Ye, G. et al. Quercetin alleviates neuropathic pain in the rat CCI model by mediating AMPK/MAPK pathway. J. Pain Res. 14, 1289–1301 (2021), PMID: 34040433, PMCID: PMC8141401.
Wang, J. Y. et al. Mechanisms underlying antidepressant effect of transcutaneous auricular vagus nerve stimulation on CUMS model rats based on hippocampal α7nAchR/NF-κB signal pathway. J. Neuroinflammation 18, 291 (2021), PMID: 34920740, PMCID: PMC8680337.
Trunnell, E. R. & Carvalho, C. The forced swim test has poor accuracy for identifying novel antidepressants. Drug Discov. Today 26, 2898–2904 (2021). PMID: 34390862.
Xiao, X., Ding, M. & Zhang, Y. Q. Role of the anterior cingulate cortex in translational pain research. Neurosci. Bull. 37, 405–422 (2021). PMID: 33566301, PMCID: PMC7954910.
Pagliusi, M. Jr & Gomes, F. V. The role of the rostral ventromedial medulla in stress responses. Brain Sci. 13, 776 (2023), PMID: 37239248, PMCID: PMC10216809.
Ni, H. et al. Upregulation of LncRNA71132 in the spinal cord regulates hypersensitivity in a rat model of bone cancer pain. Pain 164, 180–196 (2023). PMID: 35543644.
Chen, Q. & Heinricher, M. M. Shifting the balance: how top-down and bottom-up input modulate pain via the rostral ventromedial medulla. Front. Pain Res. (Lausanne) 3, 932476 (2022), PMID: 35836737, PMCID: PMC9274196.
Leonard, B. & Maes, M. Mechanistic explanations how cell-mediated immune activation, inflammation and oxidative and nitrosative stress pathways and their sequels and concomitants play a role in the pathophysiology of unipolar depression. Neurosci. Biobehav. Rev. 36, 764–785 (2012).
Maes, M., Galecki, P., Chang, Y. S. & Berk, M. A review on the oxidative and nitrosative stress (O&NS) pathways in major depression and their possible contribution to the (neuro)degenerative processes in that illness. Prog. Neuro-Psychopharmacol. Biol. Psychiatry 35, 676–692 (2011).
Fukuhara, K. et al. Intracerebroventricular 4-methylcatechol (4-MC) ameliorates chronic pain associated with depression-like behavior via induction of brain-derived neurotrophic factor (BDNF). Cell. Mol. Neurobiol. 32, 971–977 (2012).
Kim, H. et al. Brain indoleamine 2,3-dioxygenase contributes to the comorbidity of pain and depression. J. Clin. Invest. 122, 2940–2954 (2012).
Yang, R. et al. Pinocembrin inhibits P2X4 receptor-mediated pyroptosis in hippocampus to alleviate the behaviours of chronic pain and depression comorbidity in rats. Mol. Neurobiol. 59, 7119–7133 (2022). PMID: 36088500.
Schmitt, M. et al. Colon tumour cell death causes mTOR dependence by paracrine P2X4 stimulation. Nature 612, 347–353 (2022). PMID: 36385525, PMCID: PMC7613947.
Lou, Y. et al. Association of serum chemokine ligand 21 levels with asthma control in adults. Clinics (Sao Paulo) 76, e1713 (2021), PMID: 33503170, PMCID: PMC7798131.
Ikeda, H., Takasu, S. & Murase, K. Contribution of anterior cingulate cortex and descending pain inhibitory system to analgesic effect of lemon odor in mice. Mol. Pain 10, 14 (2014).

No competing interests reported.

WesternBlotimages.docx

Download PDF

Editor assigned by journal
11 Jul, 2024
Submission checks completed at journal
11 Jul, 2024
First submitted to journal
10 Jul, 2024

You are reading this latest preprint version

Possible mechanisms underlying the involvement of microglial P2X4 receptors in the comorbidity of pain and depression

Status:

Version 1

Abstract

Figures

Introduction

Methods

1.Ethics statement

2.Materials and instruments

3. Experimental animals and grouping

4. Methods and procedures

(1) Establishment of CCI model

(2) Establishment of CUMS model

(3) Thermal withdrawal latency (TWL)

(4) Mechanical withdrawal threshold (MWT)

(5) Forced swim test

5. Immunohistochemical fluorescence detection

6. Immunowestern blot detection

7. Statistical analysis

Results

1. Changes of pain threshold in each group

2. Forced swimming test results

3. Detection of cell membrane receptors (P2X4 receptors) in RVM

4. Expression of 5-HT in dorsal horn of spinal cord

Discussion

Declarations

Consent for publication:

Competing interests

Authors' information

Funding:

Author Contribution

Acknowledgements

Data Availability

References

Additional Declarations

Supplementary Files

Status:

Version 1